SOP for Microbial Limit Test by Pour Plate Method

PharmaInfo January 09, 2024
PURPOSE
To lay down the procedure for microbial testing of Raw material and Finished Products, to determine the microbial load and confirm the absence of pathogens.

SCOPE
This Procedure is applicable for Microbial limit tests in raw materials, in-process, and finished products in the microbiology section at PharmaInfo Ltd.

RESPONSIBILITY
Microbiologist / Officer: To follow the laid down procedure.
Microbiology head: All over responsibility

DEFINITION
The estimation of the number of viable aerobic microorganisms present and for freedom from designated microbial species in pharmaceutical articles of all kinds from raw material to the finished forms. It may be qualitative and quantitative or both.


PROCEDURE

Media:


Preliminary Testing:
Depending on the nature of the preparation being examined, dilute, dissolve, suspend or emulsify it in a suitable liquid. Eliminate any antimicrobial properties of the preparation by dilution, neutralization or filtration. Suitable neutralizers such as Soya Lecithin, Polysorbate, Tween 80, Glycine, and Thiosulfate are been added to neutralize certain interfering substances. They may be added to the chosen diluents or medium preferably before sterilization. If neutralizers / in activators are used for this purpose their efficacy and non-toxicity versus microorganisms must be demonstrated by carrying out a blank with neutralizer and without product.

Sample Quantity used for the Test
  • Unless otherwise directed, use 10g or 10 ml of the product to be examined.
  • The amount to be tested may be reduced for active substances that will be formulated in the following conditions: the amount per dosage unit (e.g., tablet, capsule) is less than or equal to 1 mg, or the amount per g or ml (for preparations not presented in dose units) is less than 1 mg. In these cases, the amount of sample to be tested is not less than the amount present in 10 dosage units or 10g or 10 ml of the product.
  • For materials used as active substances where the sample quantity is limited or batch size is extremely small (i.e., less than 1000 ml or 1000 g), the amount tested shall be 1 % of the batch unless a lesser amount is prescribed or justified and authorized.
  • For products where the total number of entities in a batch is less than 200 (e.g., samples used in clinical trials), the sample size may be reduced to two units, or one unit if the size is less than 100.
  • Select the sample(s) at random from the bulk material or from the available containers of the preparation. To obtain the required quantity, mix the contents of a sufficient number of containers to provide the sample.


Sample Receiving and Preparation
  • After receiving the sample in the microbiology section, record the sample details in the finished products and raw material register. The method for sample preparation depends on the physical characteristics of the product to be tested. If none of the procedures described below can be demonstrated to be satisfactory, a suitable alternative procedure must be developed.


Total aerobic microbial count (TAMC) and Total yeast and mold count (TYMC):

The preparation of the sample is as follows.

Water soluble products:
  • Dissolve or dilute 10g or 10 ml of the product to be examined in buffered sodium chloride-peptone solution pH 7.0 or in another suitable medium to make up the volume to 100ml.
  • If the product is known to have antimicrobial activity, an inactivating agent shall be added to the diluents, if necessary adjust the pH to about pH 7.0 and prepare further serial tenfold dilution using the same diluents.

Non-fatty products insoluble in water:
  • Suspend 10g or 10 ml of the product to be examined in buffered sodium chloride-peptone solution pH 7.0 or in another suitable liquid and make up the volume to 100ml.
  • A suitable surface-active agent such as 1 g / liter of polysorbate 80 may be added to assist the suspension of poorly insoluble in water substances.
  • If the product is known to have antimicrobial activity, an inactivating agent shall be added to the diluents, if necessary adjust the pH to about pH 7.0 and prepare further serial tenfold dilution using the same diluents.

Fatty Products:
  • Homogenize 10g or 10 ml of the product in isopropyl myristate or specified, with 5 g of sterile polysorbate 20 or polysorbate 80.
  • If necessary, heat to not more than 40° C. Mix carefully while maintaining the temperature in a water bath.
  • Add 85ml of buffered sodium chloride peptone solution pH 7.0 or any other suitable medium that does not have any antimicrobial agent heated to not more than 40°C.
  • Maintain this temperature for the shortest time necessary for the formation of emulsion and in any case for not more than 30 min, if necessary adjust the pH to about pH 7.0 and prepare further serial tenfold dilution using the same diluents.

If inhibitory substances are present in the sample used in the activator as per Table given below:

Pour plate method:
  • Take 2 Pre-sterilized plates and pour out 1ml of sample (Solution A) in each plate.
  • Add about 15-20ml of sterilized, cooled (40-45°C) Soybean casein digest agar medium.
  • Swirl the plates clockwise and anticlockwise to mix the sample with the media.
  • Incubate the plate in an inverted position at 30-35° C for 5 days.
  • Repeat this procedure with 2 plates of sterile Sabouraud Chloramphenicol agar (SCA).
  • Incubate these SCA plates in an inverted position at 20-25°C for 5 days.
  • At the end of all sample analyses, perform the negative control test and positive control test for the same media used in the sample analysis.
  • For negative control, Aseptically and separately transfer 1.0 ml of sterilized diluent solution into sterile Petri plates and add 15-20 ml molten soybean casein digest agar and Sabouraud chloramphenicol Agar Medium in separate Petri plates, maintained at not more than 45°C, swirl the plate to mix sample. Allow agar to solidify. Invert the Petri dishes and incubate the Soyabean Casein Digest Agar plate at 30-35°C for 3 to 5 days & Sabouraud Dextrose Agar plates at 20-25°C for 5 to 7 days.


  • For positive control, aseptically add 1.0 ml of 10-100 cfu/ml of culture inoculums in a sterile petri plate and add 15-20 ml molten soybean casein digest agar and Sabouraud Dextrose chloramphenicol Medium in separate Petri plates, maintained at not more than 45°C, swirl the plate to mix sample. Allow agar to solidify. Invert the Petri dishes and incubate the Soyabean Casein Digest Agar plate at 30-35°C for 3 to 5 days & Sabouraud Dextrose Agar plates at 20-25°C for 5 to 7 days.
  • Total aerobic microbial count / g or ml = No. of colony obtained x dilution factor/volume of sample taken
  • Total yeast and mold count / g or ml = No. of colony obtained x dilution factor/volume of sample taken.
  • Interpretation of The Results:
  1. The total aerobic microbial count (TAMC) is considered to be equal to the number of cfu found using Soybean Casein Digest Agar; if colonies of fungi are detected on this medium, they are counted as part of TAMC. The total combined yeasts and molds count (TYMC) is considered to be equal to the number of cfu found using Sabouraud Chloramphenicol Agar; if colonies of bacteria are detected on this medium, they are counted as part of TYMC.
  2. If the result of the enumeration is negative, it should be reported as "not detected for a defined unit" or "less than the detection limit for a defined unit". The result should not be
  3. If the result of the enumeration is negative, it should be reported as "not detected for a defined unit" or "less than the detection limit for a defined unit". The result should not be given as "zero for a defined unit" unless it is a regulatory requirement. Qualitative test results should be reported as "detected/not detected/absent in, a defined quantity or volume". They may also be expressed as "less than a specified number of organisms for a defined unit" where the specified number of organisms exceeds the detection limit of the method and this has been agreed with the client. In the raw data, the result should not be given as zero for a defined unit unless it is a regulatory requirement. A reported value of "0" may be used for data entry and calculations or trend analysis
  4. Reporting of Total Aerobic Microbial Counts! Total Combined Yeasts and Molds Count: After incubation count the number of colonies present in each dilution. Take the arithmetic average of the counts and calculate a number of cfu/gm of the sample tested.


Test for Specified Microorganism:

Escherichia coli:
  • Pipette out 10ml of the product from solution (A) to be examined and inoculate 10ml or the quantity corresponding to 1 g to 90 ml of soyabean casein digest broth (Solution B), homogenize and incubate at 30-35ºC for 18-24 hrs.


  • Negative Control: Use 10 ml of the same diluents used at the time of sample preparation to inoculate a suitable amount of Soyabean casein digest Broth and mix and incubate at 30-35ºC for 18-24 hrs.
  • Shake the container, transfer a quantity of the contents corresponding to 1g of the product to be examined to 100ml of Mac-Conkey broth, and incubate at 42-44ºC for 24 hrs.
  • Negative Control: Shake the negative control container, transfer 1 ml of Soyabean Casein Digest Broth to 100 ml of MacConkey Broth, and incubate at 42°C to 44°C for 24 to 48 hrs. Subculture on a plate of MacConkey Agar at 30°C to 35°C for 18 to 72 hours.
  • Subcultures on the plate of Mac-Conkey agar and incubate at 30-35ºC for 18-72 hrs.
  • Growth of red, non-mucoid colonies of gram-negative rods indicates the possible presence of E. coli.
  • If no growth of microorganisms is detected, the product passes the test.
  • Confirmative test: Indole Test: Take a red, non-mucoid colony of Gram –ve rods and add to a tube containing 5ml of 0.1% peptone water. Incubate at 42-44 °C for 24 hours. Add 0.5 ml of Kovac’s reagent, shake well, and allow to stand for 1 minute. If a red color is produced in the reagent layer, indole is present. For negative control use the sterile diluents instead of the suspected colony and then follow the section.

Salmonella:
  • Pipette out 10ml of the product from solution (A) to be examined and inoculate 10ml or the quantity corresponding to 1 g to 90 ml of soyabean casein digest broth (Solution B), homogenize, and incubate at 30-35ºC for 18- 24 hrs.
  • After 18-24 hours, shake the tube and transfer 0.1ml of solution B to tubes containing, 10ml of Rappaport vassiliadis salmonella enrichment broth medium.
  • Negative Control: Use 10 ml of the same diluents used at the time of sample preparation to inoculate a suitable amount of Soyabean casein digest Broth and mix.
  • Mix and incubate at 30-35°C for 18-24 hours.
  • Subculture on Xylose lysine deoxycholate agar.
  • Incubate at 30-35ºC for 18-48 hrs.
  • The growth of well-developed, red colonies, with or without a black center indicates the presence of Salmonella.
  • If no growth of microorganisms is detected, the product passes the test.
  • Confirmative test: Transfer separately a few of the suspect colonies to triple sugar iron agar slant tubes, using surface and deep inoculation, and incubate at 30-35ºC for 24 hrs. The formation of red slant and yellow butt and usually the formation of gas with or without production of hydrogen sulfide in the butt indicates the presence of Salmonella.

Pseudomonas aeruginosa:
  • Pipette out 10ml of the product from solution (A) to be examined and inoculate 10ml or the quantity corresponding to 1g to 90ml of soyabean casein digest broth (Solution B), homogenize and incubate at 30-35ºC for 18- 24 hrs.
  • Subculture on a plate of Cetrimide agar and incubate at 30- 35ºC for 18-72 hrs.
  • Negative Control: Shake the negative control container and subculture on a plate of Cetrimide Agar, and incubate at 30°C to 35°C for 18 to 72 hours.
  • The growth of generally greenish colonies, greenish under fluorescent UV light, and gram-negative rods indicates the presence of P. aeruginosa.


  • If no growth of microorganisms is detected, the product passes the test.
  • Confirmative test: Transfer colonies individually using an inoculating wire onto an Oxidase test disc (N N-dimethyl-p-phenylene diamine oxalate). The test is positive if a purple color is produced within 1 minute.

Staphylococcus aureus:
  • Pipette out 10ml of the product from solution (A) to be examined and inoculate 10ml or the quantity corresponding to 1g to 90ml of soyabean casein digest broth (Solution B), homogenize, and incubate at 30-35ºC for 18-24 hrs.
  • Subculture on a plate of Mannitol salt agar and incubate at 30-35ºC for 18-72 hrs.
  • Negative Control: Shake the negative control container and Subculture on a plate of Mannitol Salt Agar, and incubate at 30°C to 35°C for 18 to 72 hours
  • Yellow/white colonies with yellow zones of gram-positive cocci; indicate the presence of S. aureus.
  • If no growth of microorganisms is detected, the product passes the test.
  • Confirmative test: (Coagulase Test): Transfer the suspected colony to the tube containing 0.5ml of mammalian, preferably rabbit or horse plasma. Incubate at 35°C examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. If no coagulation in any degree is observed the sample meets the requirements of the test for the absence of Staphylococcus aureus. The product passes the test if colonies of the type described above do not appear on Mannitol salt Agar or if the confirmatory biochemical tests are negative. For negative control use sterile Buffered Sodium Chloride-Peptone Solution instead of suspected colonies in 0.5 ml of Rabbit or Horse Plasma with or without additives and follow section.


Bile Tolerant Gram Negative Bacteria:
  • Aseptically add 10g of substance to 100ml of Buffered Sodium Chloride - Peptone Solution pH 7.0 (Solution A).
  • Inoculate 10ml of solution A into 90ml of soyabean casein digest medium (Solution B1).
  • Incubate the soyabean casein digest medium (Solution B1) at 20º to 25°C for a time sufficient to resuscitate the bacteria but not sufficient to encourage the multiplication of the organisms (2-5 hours).
  • Transfer 10ml of solution B1 to 90ml of soyabean casein digest medium (Solution B2).
  • Shake the tube (Solution B2) and transfer 10ml (0.1g or 0.1ml), 1ml (0.01g or 0.01ml) and 0.1ml (0.001g or 0.001ml) of the sample to 100ml of Enterobacteria enrichment broth Mossel.
  • Incubate at 30-35°C for 24-48 hrs. After incubation shake the container, Subculture on plates of violet-red bile Glucose Agar.
  • Negative Control: Use 10 ml of the same diluents used at the time of sample preparation to inoculate a suitable amount of Enterobacteria Enrichment Broth Mossel and incubate at 30°C to 35°C for 24 to 48 hours. After incubation shake the container, Subculture on plates of violet-red bile Glucose Agar. Incubate the plates at 30°C to 35°C for 18 to 24 hours.
  • The product complies with the test if there is no growth of colonies observed on the plates.
  • Quantitative Evaluation: Inoculate suitable quantities of Enterobacteriaceae Enrichment Broth-Mossel with quantities of the solution, prepared in section 5.7.1.1 and/or dilutions of it, containing 0.1 g, 0.01 g and 0.001 g (or 0.1 ml, 0.01 ml, and 0.001 ml) of the product to be examined. Incubate at 30°C to 35°C for 24 to 48 hours. Subculture each of the cultures on a plate of Violet Red Bile Glucose Agar. Incubate at 30°C to 35°C for 18 to 24 hours


Interpretation of Results:
  • The growth of well-developed, generally red or reddish colonies of gram-negative bacteria constitutes a positive result. Note the smallest quantity of the preparation that gives a positive result and the largest quantity that gives a negative result. Determine from Table -I the probable number of bacteria. 
  • After incubation subcultures on plates of violet red bile glucose agar by means of inoculating loop and incubate at 30-35ºC for 18-24 hrs.


Clostridia:
  • Aseptically add 10g of the substance to 100 ml of buffered sodium chloride–peptone solution pH 7.0 (Solution A).
  • Take 2 equal portions corresponding to not less than 1g or 1 ml of the product to examine.
  • Heat 1 portion at 80°C for 10 minutes and cool rapidly (I)
  • Do not heat the other portion. (II)
  • Transfer 10ml of (I) & (II) containers containing 100ml of reinforced medium for clostridia.
  • Incubate under anaerobic conditions at 30º-35°C for 48 hours.
  • Negative Control: Shake the negative control container inoculate in 100 ml reinforced medium and incubate at 30°C to 35°C for 48 hours anaerobically.
  • After incubation subcultures from each tube to Columbia agar and incubate at 30º - 35°C for 48 hours.
  • Confirmative test: Catalase test: Take the suspected colony on the loop and transfer in a tube containing hydrogen peroxide. The effervescent reaction in hydrogen peroxide indicates positive. Upon examination, the occurrence of anaerobic growth of gram-positive rods giving the negative catalase reaction indicates the presence of clostridia. If no anaerobic growth of rod microorganisms is detected on Columbia agar or the catalase test is positive, the product complies with the test.

Candida albicans:
  • Aseptically add 10g of the substance to 100 ml of buffered sodium chloride-peptone Solution pH 7.0 containing 0.5% Polysorbate 80 (Solution A).
  • Inoculate 10.0ml of Solution A to 100ml of Sabouraud -dextrose broth (Solution B3).
  • Incubate the Sabouraud-dextrose broth (SDB) at 30º-35°C for 72 hours.
  • Negative Control: Shake the negative control container and inoculate in 100 ml Sabouraud -dextrose broth incubate at 30°C to 35°C for 72 hours.
  • After 3 days, shake the tube (Solution B3) and by means of inoculating loop streak a portion of Sabouraud Dextrose agar medium.
  • Incubate Sabouraud Dextrose agar plate at 30º-35ºC for 24-48 hours.
  • Gram-positive white colonies indicate the presence of C. albicans.

Shigella
  • Pipette out 10ml of the product from solution (A) to be examined and inoculate 10ml or the quantity corresponding to 1 g to 90 ml of soybean casein digest broth (Solution B), homogenize, and incubate at 30-35ºC for 18- 24 hrs.
  • Negative Control: Shake the negative control container and inoculate in 100 ml GN broth incubate at 30°C to 35°C for 18-24 hours.
  • After incubation shake the growth and transfer 1 ml to 100 ml of GN Broth Medium 11) and incubate at 30° to 35° for 24 to 48 hours.
  • Subculture on a plate of Xylose lysine deoxycholate medium (Medium 12). Incubate at 30° to 35° for 24 to 48 hours.
  • A red-colored translucent colony without a black center indicates the possibility of the presence of Shigella.
  • If a colony is found then perform an indole test for confirmation:
  • If there is no growth of such colonies or if identification tests are negative, it indicates an absence of Shigella.
  • The test is not valid unless the positive control indicates the positive result for a respective organism.
  • Limits: As per specifications.

Precaution:
The LAF bench should be validated.
All glassware should be dry and sterilized.
The autoclave should be validated.
Use positive culture carefully

ABBREVIATION
CPLR: Concept Pharmaceuticals Ltd Roorkee
SOP: Standard Operating Procedures
QA: Quality Assurance
QC: Quality Control
MIC: Microbiology
TAVC: Total Aerobic Viable Count
TAMC: Total Aerobic Microbial Count
TYMC: Total Yeast and Mould Count


REFERENCE
In-House, IP, BP, USP

ANNEXURE

REVISION HISTORY
Nil

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